1. Field of the Invention
The present invention relates to a method for analysis/inspection of a nucleic acid and a method for diagnosis using a nucleic acid as well as DNA primer sets for use therein.
2. Description of the Related Art
Demand of DNA for use in, e.g., diagnosis of disease has been increasing. Such diagnosis typically includes a method for probe test in which a DNA probe having a complementary base sequence to a target DNA is designed to see if the DNA probe hybridizes to the target DNA, and a method for obtaining sequencing information on DNA fragments produced by PCR amplification of a particular region in the base sequence of a target DNA, using two DNA probes (primers). These methods are useful for DNA up to a few DNA fragments but unsuitable for detection of DNA having a vast number of DNA fragments or for evaluation of a long DNA. However, DNAs and genes work together in vivo in association with each other so that there is a strong desire to evaluate chromosomes or all DNAs therein collectively as a whole. Where DNA functions in vivo, DNA information is initially transcribed onto mRNA, in which a protein is synthesized to make a living organism function. Noting this function, an attempt has been made in, e.g., the cDNA project to which attention has been brought in the genome project, to explore the function of the living organism based on information of the kind and amount of cDNA by producing cDNA from mRNA.
According to the cDNA project, cDNAs complementary to mRNAs expressed in cells are separated for sequencing of each cDNA, whereby frequency of the base sequence of cDNA appearing in one tissue is determined. For the determination, cDNAs are converted from mRNAs (in which various cDNAs are present in admixture) and the desired cDNAs are cloned. Escherichia coli with the cDNAs is spread over agar medium and cultured to obtain colonies. The respective colonies contain any one of the desired cDNAs. After the cDNA is collected from the corresponding colony, its base sequence is determined to identify the kind of cDNA. During the course of identifying the kind of cDNA in each colony in such a manner, the same kind of cDNA is detected. When a particular cDNA is present in a larger amount in one tissue, it means that the cDNA corresponds to a gene strongly expressed in that tissue, indicating its appearance with high frequency in the colony. Thus, cDNA sequencing is performed in a number of colonies to determine what cDNA appears how frequently (K. Murakawa et al., Genomics, 23, 379-389 (1994)).
On the other hand, an attempt has also been made to effect DNA diagnosis, noting the genome (all chromosomal DNAs) or a particular chromosome as a whole, which is called gene scanning or restriction landmark genome scanning (Y. Hayashizaki et al., DNA Polymorphism, 3, 10-15 (1995), published by Toyo Shoten). In the gene scanning, DNA is digested with a first restriction enzyme (e.g., 8-base recognition restriction enzyme such as Not I, etc., with which DNA is digested by once per 64 kb in average), and a radioisotope-tagged or fluorophore-tagged nucleotide is ligated with the digestion site, which is then subjected to electrophoresis on agarose gel toward a first direction. After separating the migrated pattern, the DNA fragment on the agarose gel is further digested with a second restriction enzyme (e.g., 4-base recognition restriction enzyme with which DNA is so digested by once per 256 bases in average), which is again subjected to electrophoresis toward a second direction intersecting to the first direction to obtain 2-dimensional migration pattern. The 2-dimensional migration pattern is utilized as a fingerprint to examine the full length DNA. Since DNA in cells with abnormality such as cancer, etc. has a different 2-dimensional pattern from DNA in normal cells, an attempt has been made to utilize the difference for diagnosis. However, it is the actual situation that there is no effective method to specify the location of abnormality in a long DNA.
As described above, it is of particular importance for understanding of the function of DNA displayed in cells to analyze a long DNA or appreciate and inspect the entire profile of a sample containing a plurality of DNAs for early identification of disease. However, any good measure is unknown in the prior art. Speaking of conventional cDNA analysis technology, it is required to determine a large number of base clones in a sample, resulting in much labor and time so that a problem encounters that conventional cDNA analysis is not practical for applying its technology to various samples. Furthermore, a method using conventional DNA probes is only available for single analysis of, at most, several to several tens of DNAs but is unsuitable for inspection of cDNAs or DNA fragments amounting to several hundreds to even several ten hundreds. The method further involves an additional problem that it is not available for inspection of such a long DNA that abnormality cannot be located.
On the other hand, DNA having a long base length can be analyzed by conventional gene scanning. However, the gene scanning also involves problems that a large volume of sample DNA is required, a considerable amount of a second restriction enzyme is required to digest the DNA fragment separated toward the first direction through electrophoresis, the mobility of each DNA fragment in the 2-dimensional migration pattern is not always quantitative in the two directions of electrophoresis to fail accurate determination of the length in each DNA fragment separated by 2-dimensional electrophoresis, resulting in difficulty to use as a database as fingerprints.